A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

Trubitsina, Liubov I.; Trubitsin, Ivan V.; Lisov, Alexander V.; Gabdulkhakov, Azat G.; Zavarzina, Anna G.; Belova, Oxana V.; Larionova, Anna P.; Tishchenko, Svetlana V.; Leontievsky, Alexey A.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s–1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s–1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

REDUCTION potential; LACCASE; AFFINITY chromatography; HUMIC acid; SODIUM azide; ORGANIC compounds; SODIUM fluoride

Biochemistry (00062979), 2023, Vol 88, Issue 10, p1658

0006-2979

Academic Journal

10.1134/S0006297923100188